中华实验眼科杂志
中華實驗眼科雜誌
중화실험안과잡지
CHINESE JOURNAL OF EXPERIMENTAL OPHTHALMOLOGY
2011年
8期
681-684
,共4页
王兴利%马强%曾玉晓%何建荣%王一
王興利%馬彊%曾玉曉%何建榮%王一
왕흥리%마강%증옥효%하건영%왕일
α晶状体蛋白%生物素化%视网膜神经节细胞
α晶狀體蛋白%生物素化%視網膜神經節細胞
α정상체단백%생물소화%시망막신경절세포
α-Crystallin%Biotinylated%Retinal ganglion cells
背景 视神经损伤后无法有效再生,而近年研究发现,α晶状体蛋白能显著促进视网膜神经节细胞(RGCs)的存活及轴突有效再生,但其分子机制尚不清楚.目的 研究外源性α晶状体蛋白与RGCs的结合部位.方法 从2只2 d龄Long Evans大鼠视网膜中分离并原代培养RGCs,应用thy1.1和cy3抗体荧光染色技术对培养的RGCs进行鉴别并在荧光显微镜下计数RGCs阳性率.对外源性的α晶状体蛋白生物素化后用直接显色法进行鉴定,并用胰岛素实验对其分子伴侣活性进行测定,明确α晶状体蛋白生物素化成功并具有分子伴侣活性后与原代培养的RGCs共孵育,再与荧光标记的亲和素进行反应,在激光共焦显微镜下观察外源性α晶状体与RGCs的结合部位.结果 原代培养的RGCs阳性率为94%;生物素化α晶状体蛋白直接显色法鉴定整体显色强,其A450值随生物素化的α晶状体蛋白的浓度下降而下降,提示α晶状体蛋白生物素化成功;生物素化α晶状体蛋白的分子伴侣活性明显,且生物素化前后活性无明显改变;生物素化α晶状体蛋白与RGCs共孵育及荧光染色后,激光共焦显微镜下与生物素化α晶状体蛋白共孵育的RGCs细胞膜和轴突均可见红色荧光,细胞质和细胞核未见荧光染色.对照组RGCs未见荧光染色.结论 外源性的α晶状体蛋白特异性地结合在RGCs细胞膜上,提示外源性的α晶状体蛋白通过与RGCs细胞膜特异性结合而发挥相应的生物学功能,但其结合方式及作用机制需进一步研究.
揹景 視神經損傷後無法有效再生,而近年研究髮現,α晶狀體蛋白能顯著促進視網膜神經節細胞(RGCs)的存活及軸突有效再生,但其分子機製尚不清楚.目的 研究外源性α晶狀體蛋白與RGCs的結閤部位.方法 從2隻2 d齡Long Evans大鼠視網膜中分離併原代培養RGCs,應用thy1.1和cy3抗體熒光染色技術對培養的RGCs進行鑒彆併在熒光顯微鏡下計數RGCs暘性率.對外源性的α晶狀體蛋白生物素化後用直接顯色法進行鑒定,併用胰島素實驗對其分子伴侶活性進行測定,明確α晶狀體蛋白生物素化成功併具有分子伴侶活性後與原代培養的RGCs共孵育,再與熒光標記的親和素進行反應,在激光共焦顯微鏡下觀察外源性α晶狀體與RGCs的結閤部位.結果 原代培養的RGCs暘性率為94%;生物素化α晶狀體蛋白直接顯色法鑒定整體顯色彊,其A450值隨生物素化的α晶狀體蛋白的濃度下降而下降,提示α晶狀體蛋白生物素化成功;生物素化α晶狀體蛋白的分子伴侶活性明顯,且生物素化前後活性無明顯改變;生物素化α晶狀體蛋白與RGCs共孵育及熒光染色後,激光共焦顯微鏡下與生物素化α晶狀體蛋白共孵育的RGCs細胞膜和軸突均可見紅色熒光,細胞質和細胞覈未見熒光染色.對照組RGCs未見熒光染色.結論 外源性的α晶狀體蛋白特異性地結閤在RGCs細胞膜上,提示外源性的α晶狀體蛋白通過與RGCs細胞膜特異性結閤而髮揮相應的生物學功能,但其結閤方式及作用機製需進一步研究.
배경 시신경손상후무법유효재생,이근년연구발현,α정상체단백능현저촉진시망막신경절세포(RGCs)적존활급축돌유효재생,단기분자궤제상불청초.목적 연구외원성α정상체단백여RGCs적결합부위.방법 종2지2 d령Long Evans대서시망막중분리병원대배양RGCs,응용thy1.1화cy3항체형광염색기술대배양적RGCs진행감별병재형광현미경하계수RGCs양성솔.대외원성적α정상체단백생물소화후용직접현색법진행감정,병용이도소실험대기분자반려활성진행측정,명학α정상체단백생물소화성공병구유분자반려활성후여원대배양적RGCs공부육,재여형광표기적친화소진행반응,재격광공초현미경하관찰외원성α정상체여RGCs적결합부위.결과 원대배양적RGCs양성솔위94%;생물소화α정상체단백직접현색법감정정체현색강,기A450치수생물소화적α정상체단백적농도하강이하강,제시α정상체단백생물소화성공;생물소화α정상체단백적분자반려활성명현,차생물소화전후활성무명현개변;생물소화α정상체단백여RGCs공부육급형광염색후,격광공초현미경하여생물소화α정상체단백공부육적RGCs세포막화축돌균가견홍색형광,세포질화세포핵미견형광염색.대조조RGCs미견형광염색.결론 외원성적α정상체단백특이성지결합재RGCs세포막상,제시외원성적α정상체단백통과여RGCs세포막특이성결합이발휘상응적생물학공능,단기결합방식급작용궤제수진일보연구.
Background There is no effective method to regenerate the optic nerve after injury. It has been recently reported that α-crystallin could promote the survive rate and axon regeneration of retinal ganglion cells (RGCs) effectively. However,the molecular mechanism is not clear. Objective This study was to identify the site of RGCs where the exogenous α-crystallin bind to. Methods RGCs was isolated from retinas of two 2-day-old Long Evans rats and primarily cultured. The positive rate of the RGCs was assessed by counting the number of positive cells for fluorescently-labeled thy1. 1 and cy3 under the fluorescence microscope. The biotinylated exogenous α-crystallin was evaluated by direct coloration and the activity of molecular chaperones was measured by insulin test.After identifying the success of biotinylation along with the activity of molecular chaperones,biotinylated α-crystallin was co-incubated with RGCs and the cells then were reacted to fluorescently labeled avidin for the observation of binding site of exogenous α-crystallin under the laser confocal microscope. Results RGCs of 94% were survived through primary culture. The coloration of biotinylated α-crystallin labeled by the direct coloration method was more intensive, and the value of A450 descended as the decrease of biotinylated α-crystallin concentration,indicating that the α-crystallin was biotinylated successfully. The activity of molecular chaperones of biotinylated α-crystallin was significantly strong but no significant change after being biotinylated after co-incubation of RGCs with biotinylated α-crystallin. Laser confocal microscope examination revealed that co-incubated RGCs with biotinylated α-crystallin showed the red fluorescence on membrane and axon of RGCs rather than cytoplasm and nucleus. The absent response was seen in the control group. Conclusion Exogenous α-crystallin can specifically combine with the membrane of RGCs to play the biological function,but its binding mode and mechanism need further study.
